Nickel is an essential element for growth of many microorganisms, as this element is a component of enzymes involved in ureolysis, H2 metabolism, methane production and carbon monoxide utilization. Among the bacteria that utilize nickel for one of these processes, many are bacteria that contain human pathogenic representatives within the genus. Nickel metabolism in general is also of health concern due to its toxic effects on humans. Nevertheless, our knowledge of nickel metabolism is poor. For example, no intracellular Ni-metabolism steps have been identified, even though it is likely that sequential enzymatic nickel-metabolizing processes occur. This proposal is aimed at elucidating the routes of nickel binding, internalization, metabolism, and incorporation into a bacterial enzyme (hydrogenase) in the bacterium Bradyrhizobium japonicum. The specific aims are to: 1. Purify and characterize a putative nickel storage protein; 2. Determine the number and nature of extracellular and intracellular nickel binding/metabolizing proteins; 3. Determine if nickel can transcriptionally regulate the synthesis of a nickel-containing enzyme; 4. Isolate and characterize mutants in specific nickel-metabolism genes; 5. Isolate genes involved in nickel metabolism and nickel storage to determine their organization, and determine the occurence of these genes in other nickel-utilizing bacteria. The approach included bacterial physiology and biochemistry to determine the pathways of nickel sequestering, storage, processing and eventual insertion into the nickel-containing metalloenzyme hydrogenase. Mutants in nickel metabolism will be isolated to aid in identifying Ni-metabolism enzymes, and molecular genetic techniques will be used to isolate the genes involved. The long-term goal is to help in understanding biochemically and genetically, the number, mature, function, and ubiquity of the components (proteins, genes, other factors) involved in bacterial nickel metabolism.